To further clarify the physiological functions of intracellular enzymes catalyzing the hydrolysis of CO-NH bonds between L-amino acid residues in di-, tri-, and oligopeptides, attempts will be made to determine whether any of the peptidases we have detected in extracts of Escherichia coli K-12 (and partially characterized) are specifically involved in protein turnover. Using a well-defined in vitro coupled transcription-translation system to follow incorporation of amino acids into proteins, the effects of known "methionine aminopeptidases" on the rate of protein synthesis by strain K-12 will be investigated; a comparison also will be made of the protein-synthesizing ability of strain K-12 and "peptidase mutants." One such mutant already is available and others will be sought with detection methods based on the substrate specificities of known peptidases. The ability of strain K-12 and its peptidase mutants rapidly to degrade proteins in vivo will be compared by following the disappearance of inducible enzymes (beta-galactosidase, alkaline phosphatase) and abnormal proteins (containing amino acid analogs) and the appearance of free amino acids during from protein degradation in growing and starved cells. A comparison of the growth cycles of strain K-12 and peptidase mutants will be made to ascertain whether any specific peptidase is essential for metabolic processes associated with the initiation of logarithmic growth and with resistance to the bacteriostatic effects of leucine-containing peptides. Peptidase purification and characterization will be continued with emphasis on enzymes showing potent methionine aminopeptidase activity on a tetralysine endopeptidase identified but not characterized by Sussman and Gilvarg.